Abstract Transcription factors play crucial roles in regulation of gene expression. A deeper understanding of these proteins will facilitate development of human therapeutics because many human diseases and disorders are associated with dysfunction or abnormality of transcription factors. To regulate genes, transcription factors must first bind to their functional targets within cis-regulatory elements such as promoters and enhancers in the genome. Knowledge of the specific interactions with cis-regulatory elements is essential, but insufficient for us to completely understand how transcription factors work. Although many transcription factors recognize particular DNA sequences with high affinity, these proteins also bind to other DNA sequences with weaker affinity. The vast quantity of nonspecific sites in the genome compensates for their weak affinity, making profound overall impacts on transcription factors. Kinetic and thermodynamic efficiency for transcription factors to bind to their functional targets should be influenced strongly by prior interactions with non-target sites on genomic DNA. The overall objective in this project is to deepen our understanding of dynamic processes whereby transcription factors scan DNA, recognize particular sequences, and locate functionally important sites for regulation of genes. Using biophysical, biochemical, and cell-biological approaches, the research team of this project will pursue the following two questions: 1) How do transcription factors reach functional targets on genomic DNA? 2) How do electrostatic interactions occur between proteins and DNA? The genome contains numerous high-affinity sequences for transcription factors, but only a very small fraction of the sites are functional for gene regulation. The vast majority of these high-affinity sites serve as natural decoys that could sequester the transcription factors in nonfunctional regions. The research team will test a hypothesis that sequestration in natural decoys and alteration of their accessibility serve as controllable mechanisms that regulate efficiency in target DNA association of transcription factors. The research team will also pursue advancing the atomic-level knowledge of the DNA scanning process, focusing on the behavior of side chains crucial for DNA recognition. The PI's group recently revealed the highly dynamic nature of interfacial ion pairs and their entropic roles in protein-DNA association. The research team will further study the roles of the ion- pair dynamics in DNA recognition and scanning by transcription factors. The research team showed that chemical modifications of the intermolecular ion pairs could significantly enhance protein-DNA binding affinity. The research team will apply this knowledge toward improving synthetic decoys for transcription factors. Through these studies, this project will help improve therapeutics targeting transcription factors relevant to human diseases and disorders.